In many countries, the occurrence of clarithromycin-resistant Helicobacter pylori has become increasingly common. Most clinical resistant strains with MICs of clarithromycin exceeding 2 mg/liter are characterized by mutations in the 23S rRNA gene at positions 2058 and 2059 (Escherichia coli numbering). We isolated two stable H. pylori isolates (TS1900 and TS1776) with low-level clarithromycin resistance (MICs of 1 mg/liter and 0.5 mg/liter, respectively) that lacked these mutations and the mutation at position 2628 (E. coli numbering) reported by Fontana et al. to confer low-level resistance. By comparing the 23S rRNA gene sequence of the clarithromycin-susceptible strain ATCC 700392 with that of TS1900, we identified 13 substitutions. Transformation experiments showed that the C2611A mutation (E. coli numbering, corresponding to position C2694 of H. pylori) in the 23S rRNA gene was associated with low-level clarithromycin resistance. TS1776 also possessed this mutation. DNA sequencing analysis of 58 clinical isolates in Japan from 1996 to 2006 and the DNA Data Bank of Japan indicated that the C2611A mutation was not detected in any clarithromycin-susceptible or -resistant isolates. We suggest that the C2611A mutation may introduce a steric hindrance that prevents efficient clarithromycin binding to the ribosome, thus conferring low-level clarithromycin resistance in H. pylori.